Vitamins K hydroquinone, oxygen, CO2, and a membrane-bound carboxylase are required for the gamma-carboxylation of selected glutamate residues on preprothrombin and other precursor peptides of vitamin K-dependent proteins. The mechanism of this enzymatic reaction is still obscure. It is becoming increasingly likely, however, that free radicals are intermediates including the semiquinone of vitamin K. Preliminary evidence suggests that anti-oxidants, including vitamin E, antagonize vitamin K and increase the vitamin K requirement in animals and in man. The objective of this proposal is to study the interaction between vitamin E, other antioxidants and vitamin K. The vitamin K requirement will be determined in rats fed diets containing variable amounts of vitamin E, N,N-diphenylphenylene diamine (DPPD), ethoxyquin and butylated hydroxytoluene. In order to determine the mechanism of interaction between these antioxidants and vitamin K, their effect on the absorption and metabolism of tritium-phylloquinone and their effect on the in vitro vitamin K-dependent carboxylation system will be studied. In addition, evidence for radical intermediates in purified carboxylase preparations will be sought by electron spin resonance. At the present time there is no satisfactory method for the measurement of vitamin K in plasma. It is hoped that the application of the techniques of high pressure liquid chromatography and GLC-mass spectrometry will improve the sensitivity of the method so that plasma vitamin K levels under various conditions can be measured. It is also planned to develop a radioimmunoassay for the detection of vitamin K in plasma.